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RNA catalysis: ribozymes, ribosomes, and riboswitches
RNA catalysis: ribozymes, ribosomes, and riboswitches Scott A Strobel and Jesse C Cochrane The catalytic mechanisms employed by RNA are chemically more diverse than initially suspected. Divalent metal ions, nucleobases, ribosyl hydroxyl groups, and even functional groups on metabolic cofactors all contribute to the various strategies employed by RNA enzymes. This catalytic breadth raises intriguing evolutionary questions about how RNA lost its biological role in some cases, but not in others, and what catalytic roles RNA might still be playing in biology. Address Yale University, Department of Molecular Biophysics and Biochemistry, 260 Whitney Avenue, P.O. Box 208114, New Haven, CT 06520-8114, United States Corresponding author: Strobel, Scott A ([email protected])
Current Opinion in Chemical Biology 2007, 11:636–643 This review comes from a themed issue on Biopolymers Edited by Jennifer Kohler and Jason Chin
In this review, we will examine four examples of RNA catalysis, each of which provides a different chemical strategy used to promote a ribozyme reaction. This includes: the group I class of self-splicing introns which employs two divalent metal ions to promote RNA cleavage and ligation reactions; the Hepatitis Delta Virus ribozyme which catalyzes autolytic RNA cleavage using an essential, pKa perturbed cytidine; the glmS ribozyme which is an autolytic riboswitch that uses the small molecule metabolite glucosamine-6-phosphate (GlcN6P) as a catalytic cofactor; and the ribosomal peptidyl transferase center which does not appear to use any of these strategies, but instead forms peptide bonds with the assistance of a functional group on the tRNA substrate. The diversity of solutions employed by RNA to achieve reaction catalysis is unexpected; raising intriguing questions about the evolution and devolution of RNA catalysis. What led RNA to lose its catalytic role in some biological cases but not others, and what other catalytic functions might RNA still be playing in biology?
Available online 5th November 2007 1367-5931/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2007.09.010
RNA is well suited for its role as a purveyor of genetic information. The consistent hydrogen-bonding potential of each nucleobase is crucial for fidelity during replication, transcription, and translation. It is easy to imagine evolutionary pressures that would select for residues with such properties. This is achieved in part because none of the nucleobases have ionizable groups near neutrality. Although optimized for reliable base pairing, RNA is also able to catalyze essential biochemical reactions, including RNA processing and protein synthesis. It does this despite significant biophysical handicaps. RNA has a small repertoire of functional groups and these are embedded in a poorly constrained ribosyl-phosphate backbone heavy with negative charge. The pKas of the bases would appear to be either too low (3.5 for A and 4.2 for C) or too high (9.8 for G and 10.5 for U) for use in efficient general acid catalysis or base catalysis. As a group, RNA enzymes, or ribozymes, are less efficient catalysts relative to chemically supercharged proteins, yet in many cases ribozymes provide enough rate enhancement to have escaped replacement by protein alternatives in the evolution from an RNA World to the protein dominated world of modern biochemistry. Current Opinion in Chemical Biology 2007, 11:636–643
Group I intron — two-metal mechanism RNA splicing by the group I intron involves two symmetrical phosphoryl transfer reactions, both catalyzed by the intron itself (Figure 1). In the first reaction, an exogenous G attacks the 50 -splice site to release the 50 exon from the intron. In the second reaction, the 50 -exon attacks the 30 -splice site, at the phosphate 30 to the last G of the intron (VG), to produce ligated exons and liberate the intron. The second reaction is essentially the reverse of the first, differing only in whether exogenous G or VG is bound in the active site. An early model for group I intron catalysis was proposed by Steitz and Steitz based upon analogy to protein enzymes that catalyze phosphoryl transfer [1]. Their proposal involved two active site divalent metal ions bridging across the scissile phosphate positioned 3.9 A˚ apart, one activating the O30 nucleophile and the other activating the O30 leaving group. Although at the time no evidence for this model was provided, it was greeted enthusiastically and efforts were made to find these catalytic metals within the group I intron and other ribozymes. Biochemical analysis provided the initial evidence for metal ion participation in group I intron catalysis. Active site metal ion coordination was probed using metal specificity switch analysis. These experiments change the identity of a putative metal ligand from an oxygen to a sulfur. The electronically ‘soft’ sulfur substitution disrupts Mg2+ coordination, rendering the RNA inactive when the substitution is at a site of metal binding. Activity rescue upon addition of a ‘soft’ metal provides www.sciencedirect.com
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Figure 1
Group I intron splicing promoted by two catalytic metal ions. The second of the two-step splicing reactions is shown. The nucleophile (U-1 O30 ), scissile phosphate, leaving group (VG O30 ), and labile bond are shown in orange. Both catalytic metal ions are shown in green. A similar coloring scheme is used in all four figures where the chemically reactive groups and labile bond are shown in orange and the catalytically important groups are shown in green. (a) Active site structure adapted from Ref. [7]. (b) Reaction mechanism [7].
evidence for metal ion coordination. Single and pair wise substitutions were made throughout the group I intron active site. Consistent with the Steitz and Steitz model, the biochemical data suggested that metals coordinate the nucleophile, leaving group and scissile phosphate, but the intron also has a metal coordinating the 20 -OH vicinal to the leaving group (Figure 1). As a result, an alternative three-metal mechanism was proposed, in which none of the metals bridged between the scissile phosphate and the VG O30 leaving group [2,3]. This active site architecture differed significantly from that proposed for the two-metal model. X-ray crystal structures of the group I intron became available in the past few years. Structures of three different www.sciencedirect.com
introns in three different reaction states were reported in 2004 [4–6]. These provided three-dimensional views of the group I intron, but the RNA in each of these structures lacked at least one of the active site functional groups known to coordinate a catalytic metal ion. The structure of a catalytically active complex, including the complete intron and both exons and all metal ligands, was reported a year later (Figure 1) [7]. The overall structure of the intron was unchanged from the previous structures, but significant differences were observed within the active site. The structure revealed two well-ordered and extensively coordinated Mg2+ ions positioned 3.9 A˚ apart, both making inner sphere coordination to the scissile phosphate. Metal M1 is coordinated to the terminal O30 of the 50 -exon and the pro-RP oxygen of the scissile phosphate. Metal M2 also makes inner sphere coordination to the pro-RP oxygen of the scissile phosphate, as well as the O30 and O20 of VG. Both metals have a total of five inner sphere coordinations including interactions with phosphate oxygens within the intron. The two metals are coordinated by all of the biochemically predicted ligands, including two ligands identified within the intron. This two-metal ion structure suggests that the group I intron active site is mechanistically equivalent to a large number of protein-based phosphoryl transferases, including all known DNA and RNA polymerases. RNA enzymes and protein enzymes could not be evolutionarily related, so the equivalence of group I intron and polymerase active sites must result from convergent evolution. The intron uses the phosphate backbone in a manner equivalent to aspartate site chains in proteins for active site metal ion coordination, including one phosphate within the intron that uses both of its nonbridging oxygens to directly coordinate both active site metals. That macromolecular evolution arrived independently at the same solution in RNA and proteins implies an intrinsic catalytic capacity of the two-metal-ion catalytic architecture for phosphoryl transfer. What remains unknown is whether other RNA enzymes, such as the group II intron, RNase P, or the spliceosome, use the same catalytic strategy. Two RNase P structures were reported recently, but both complexes are of apoenzymes without the tRNA substrate [8,9]. Biochemical experiments strongly implicate metal ions in catalysis [10], but conformational changes or metal ion rearrangements might accompany substrate binding. No highresolution structural views of the group II intron or splicesome exist, however biochemical data implicate divalent metal ions in catalysis in both systems [11,12].
HDV ribozyme — nucleobase catalysis Divalent metals are not the only solution RNA has found to catalyze its reactions. The symmetry of the two-metal mechanism led to the premature conclusion that all ribozymes are metalloenzymes. Not every protein enzyme Current Opinion in Chemical Biology 2007, 11:636–643
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uses metals for phosphoryl transfer, and it is now clear that this is also true for ribozymes. For example, ribonuclease A employs two catalytic histidines to activate the nucleophile and stabilize the leaving group during the RNA cleavage reaction it promotes. Histidine, with its pKa of 6.8, is ideally suited for these roles at physiological pH. Because RNA lacks any nucleobase functional groups with a pKa near neutrality, nucleobase participation appeared to be an unlikely contributor to ribozyme chemistry. But it was then observed that several catalytic RNAs, including the HDV ribozyme, retained reasonable activity in the presence of high concentrations of monovalent ions, or other polyvalent ions that could not make inner sphere coordination to RNA ligands [13]. This suggested that alternative mechanisms must be in play. The HDV ribozyme is a small autonucleolytic RNA that shares a common chemistry with the other nucleolytic ribozymes. The 20 -OH next to the scissile phosphate acts as nucleophile to displace the O50 resulting in two RNA products, one with a 20 –30 cyclic phosphate terminus and one with a 50 -OH terminus (Figure 2). This is the same chemical reaction catalyzed by ribonuclease A. The first HDV crystal structure, of the product form of the ribozyme, showed that the 50 -OH was within hydrogen-bonding distance to the N3 of C75, an invariant residue indispensable for efficient cleavage [14]. The inactive C75U mutation could be rescued with imidazole and the pKa of the reaction appeared to track with the pKa of nucleobases substituted at the key residue [15,16]. This suggested that C75 might function as a general acid to protonate the O50 leaving group. However, subsequent crystal structures of the substrate form of the ribozyme showed that the C75 N3 is closer to the nucleophile, instead implicating C75 as a general base to deprotonate the 20 -OH [17]. This controversy was biochemically resolved in an elegant experiment showing that an enzyme with an activated leaving group no longer required this C for function [18]. These data strongly favor the original model; C75 acts as a general acid to protonate the leaving group and a divalent metal ion activates the nucleophile (Figure 2). Although C75 is not as efficient as histidine in this role because of its acidic pKa, the microenvironment of the enzyme active site appears to perturb the pKa toward neutrality. The precedent of general acid catalysis by the HDV ribozyme argues that other catalytic RNAs could employ such a strategy. Nucleobase catalysis has now been implicated in all of the autolytic ribozymes, but the diversity of the bases involved has come as a surprise. A critical A has been implicated in the VS ribozyme [19]. The crystal structure of the full-length hammerhead ribozyme reveals two critical Gs in the active site [20]. The N1 of G12 is near the O20 nucleophile and the 20 -OH of G8 is close to the O50 leaving group. The participation of G in RNA catalysis is unexpected as it has Current Opinion in Chemical Biology 2007, 11:636–643
Figure 2
Autolytic cleavage of RNA by the HDV ribozyme. Color scheme as in Figure 1. (a) Active site structure adapted from Ref. [14]. Residue A-1 was modeled into the coordinates for ease of comparison to the chemical scheme. (b) Reaction mechanism [16,18].
a alkaline pKa and the deprotonated form is anionic. It is relatively easy to envision how the highly anionic environment of an RNA could perturb a cationic pKa higher, but its harder to understand how the electrostatics of RNA could perturb an anionic pKa lower. Yet, Gs are also implicated in catalysis by the hairpin, VS and glmS (see below) ribozymes [21,22]. Proposed models for the role these Gs play in the reaction include simple substrate alignment by hydrogen bonding or proton transfer through tautomerization of the base [23,24].
Ribosomal peptide-bond formation by substrate assistance The peptidyl transferase center of the ribosome is also a ribozyme [25]. Initial mechanistic models for this reaction employed nucleobase involvement in catalysis (specifically A2451), however mutational analysis of this and www.sciencedirect.com
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other active site residues found that they contributed relatively little to the rate of peptide-bond formation [26– 28]. The effects were larger for the hydrolysis reaction that occurs at termination, but even in that reaction the largest rate decreases were still about 100-fold. Further, all crystallographic efforts to identify active site metal ions have also been unsuccessful [29]. While there is an obligate Mg2+ requirement for peptidyl transferase activity, the contribution does not appear to result from direct chemical participation. Relative to the rate in solution, the peptidyl transferase center of the ribosome provides a rate enhancement of approximately 107-fold, a contribution that has been largely attributed to entropy [30]. Beyond substrate alignment, how else might the ribosome promote this biologically essential reaction? The peptidyl transferase reaction involves two substrate tRNAs, an aminoacyl-tRNA that binds to the ribosomal A site and a peptidyl tRNA that binds to the P site. The nascent peptide in the P site is linked via an ester to the 30 -O of the terminal nucleoside of the tRNA, A76 Figure 3
(Figure 3) [31]. The reaction involves aminolysis of the P-site ester by the A-site a-amino group. The ribosome aligns the two substrates such that the amine approaches the sc face of the P-site ester, resulting in a chiral transition state with an S stereochemistry [29]. The A76 20 -OH vicinal to the P-site ester is essential for the reaction [32]. Deletion or alteration of this functional group results in a complete loss of peptidyl transferase activity (>106-fold loss of activity). Without this hydroxyl group, the rate of spontaneous ester hydrolysis is faster than the rate of peptide-bond formation. This contribution is significantly greater than that made by any of the rRNA nucleobases within the active site, though the 20 -OH of A2451 has also been shown to contribute to the reaction [33]. Hydroxyl groups are unlikely catalytic players in a reaction that appears to involve proton transfer. With a pKa of approximately 12, the 20 -OH is probably less likely than the nucleotide bases to be involved in proton transfer even if the pKa were significantly perturbed. Furthermore, there is nothing about the ribosome active site that would lead to such a perturbation. All unexplained density near the reaction center appears to be water, including one molecule positioned within the oxyanion hole [29]. Mechanistic models must therefore explain the role of the 20 -OH without invoking a significant degree of protonation or deprotonation. The ribosome positions the critical A76 20 -OH between the a-amino nucleophile and the 30 -O leaving group (Figure 3). As the amine must lose a proton and the O30 must gain one, mechanisms have been proposed that involve concerted proton transfer from the a-amine to the A76 O30 via the A76 20 -OH [29,34–37]. These mechanisms explain the need for the 20 -OH without having it assume a significant charge, consistent with its elevated pKa. Several mechanisms have been proposed that invoke this role for the 20 -OH, each differing in the extent of peptide-bond formation and the degree of leaving group dissociation in the transition state. Although the mechanisms share common themes, they make significantly different predictions regarding the nature of the transition state and the contribution the ribosome could make to catalysis. The answer will require enzymatic analysis to define the degree of bonding and distribution of charge in the transition state. Only then will it be clear exactly what transition state the ribosome stabilizes during peptidebond formation and the precise role of the 20 -OH of A76.
Peptide-bond formation promoted by the ribosome. The a-amine, carbonyl carbon, O30 leaving group and scissile bond are shown in orange. The critical 20 -OH is in green. (a) Active site structure adapted from Ref. [29]. (b) Proposed reaction mechanism [29,34]. www.sciencedirect.com
Independent of these mechanistic details, the observation that a functional group on the tRNA substrate provides a major catalytic contribution invokes an additional role for tRNAs in translation. Even before the discovery of tRNA, Crick proposed the adaptor hypothesis, which predicted a molecule capable of two functions: interaction with an Current Opinion in Chemical Biology 2007, 11:636–643
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mRNA and delivery of the correct amino acid. tRNA plays both these roles, but the adaptor model suggests a passive role for tRNA during translation. tRNA is aminoacylated by synthases and used by the ribosome for decoding, but the tRNAs are the object, rather than the subject of action in these roles. In a recent review, Woese called for a reevaluation of tRNA during translation [38], and there are now indications that tRNA does play a significant proactive role, including a spring-like distortion of the tRNA during accommodation into the A site where it provides the key functional group for peptide-bond formation to proceed [32,39].
Figure 4
GlmS ribozyme — use of a catalytic cofactor And when it appeared that all permutations for RNA catalysis had been exhausted, a ribozyme was identified that uses still another mechanistic strategy (Figure 4). The glmS riboswitch is located in the 50 -untranslated region of the gene encoding glucosamine-6-phosphate (GlcN6P) synthetase [40]. In the presence of GlcN6P, it cleaves its own mRNA, which downregulates the production of the synthetase. GlcN6P is essential for efficient cleavage activity, though other primary amines with a vicinal hydroxyl group can also activate the ribozyme in vitro, including serinol and Tris [41]. The primary amine is essential for the reaction; substitution of the amine with a hydroxyl results in complete loss of activity. Glucose-6 phosphate (Glc6P) is still able to bind to the ribozyme, but functions as a competitive inhibitor. Two possible models for the role of GlcN6P in glmS catalysis can be envisioned. In traditional riboswitches, metabolite binding induces a conformational change in the RNA that affects the transcription or translation of the gene [42]. In this model for riboswitch function, binding of the GlcN6P would stabilize the active ribozyme conformation and lead to catalysis. In the alternative model, GlcN6P binding does not affect the conformation of the RNA, but instead the critical amine on the metabolite participates directly in chemistry. Biochemical and structural data both indicate that the contribution made by GlcN6P is not conformational, but is instead chemical [40,43,44,45]. Initial inline probing experiments showed that the conformational flexibility of the RNA is unchanged upon GlcN6P binding [40]. Hydroxyl radical footprinting experiments showed that the RNA had an equivalent solvent accessible surface in both the bound and free states [43]. Consistent with these observations, independent crystal structures revealed that the RNA adopts the same conformation in the metabolite bound, inhibitor bound and free states [44,45]. Thus, glmS is not a traditional riboswitch, changing its conformation upon metabolite binding, instead it uses the chemical capability of the metabolite in catalysis. The pKa of the GlcN6P amine is 8.3, a value reasonably well suited for a role in proton transfer. This demonstrates that Current Opinion in Chemical Biology 2007, 11:636–643
Autolytic cleavage of RNA by the glmS ribozyme in the presence of GlcN6P. The color scheme is as in Figure 1 with the critical amine of GlcN6P and the N1 of G33 shown in green. (a) Active site structure adapted from Ref. [45]. (b) Proposed reaction mechanism [45]. The identity of G33 is important to the reaction, but its role remains to be fully defined.
RNA, like protein enzymes, can employ the chemical potential of small molecules to promote catalytic activity. How does GlcN6P serve as a catalytic cofactor? The authors of the initial structure of the Glc6P inhibitor bound ribozyme suggested that the amine might serve as a general base to deprotonate the O20 nucleophile [44]. Although the C2 hydroxyl is not well positioned for this role, the authors proposed that it could act through a chain of two water molecules present in the active site. The GlcN6P bound structure superimposes on the Glc6P structure, but no water molecules for proton shuttling to the O20 were observed [45]. In both structures the amine (or its hydroxyl substitute) is within hydrogenbond distance of the O50 leaving group (Figure 4). This suggests a role for the amine as a general acid to activate www.sciencedirect.com
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the leaving group. A definitive answer to this question awaits the kind of analysis that was performed on the HDV ribozyme [18]. The closest functional group to the O20 nucleophile is not on GlcN6P, but is the N1 of G33 (Figure 4). Consistent with a role in ribozyme function, mutation of G33 disrupts activity [45]. However, the precise contribution of this functional group to glmS ribozyme catalysis is also unknown. Nevertheless, the primary catalytic contribution is expected to come through the metabolic cofactor, as activation of the leaving group is the chemically difficult step in phosphoryl transfer reactions.
are fidelity, processivity, and reaction rates, why did these same parameters not lead to a protein-based system for peptide-bond formation? This has not happened, or at least hasn’t happened yet. The amino terminus of protein L27 is intriguingly close to the peptidyl transferase center as observed in the recent 70S crystal structure from Thermus thermophilus [46]. L27 is not conserved and so is not essential for protein synthesis in all organisms, but this terminal extension is essential in E. coli [47]. L27 may represent an adaptation, possibly an evolutionary intermediate between an RNA and a protein-based active site for protein synthesis.
Implications
Studies of the glmS ribozyme revealed that RNA can use small molecules as catalytic cofactors. With an expanded chemical repertoire made possible by small organic compounds, what other reactions did (or still does) RNA catalyze in biological systems? To date, riboswitches responsive to at least 10 different small molecules have been identified [42]. The metabolites include coenyzme B12 (B12), flavin mononucleotide (FMN), thiamine pyrophosphate (TPP), S-adenosyl methionine (SAM), guanine, adenine, glycine, lysine, and GlcN6P. The intriguing nature of this list is that many of the metabolites that are recognized by riboswitches are also among the most ubiquitous cofactors used by protein enzymes, particularly B12, FMN, TPP, and SAM. Protein enzymes use these cofactors and substrates for radical chemistry, oxidation– reduction reaction and carbon–carbon bond formation. To date, chemistry promoted by naturally occurring RNAs has been confined to phosphoryl transfer and transesterification. Naturally occurring RNAs bind these small molecule cofactors. The use of their chemical moieties in chemistry could significantly expand the catalytic repertoire of RNA. Ribozymes with radical, redox and methyltransferase activities were needed for metabolism in the RNA World. Which, if any of them, have persisted into modern biology remains to be determined.
The mechanisms employed by RNA to promote chemistry are substantially more diverse than once thought. They include metals, nucleobases, ribosyl groups, and even small molecule cofactors. Such observations raise questions regarding the evolution of biocatalysis and the diversity of reactions that RNA might catalyze. The observation that the group I intron has a polymeraselike active site built of RNA leads to the question: What selective pressure led to the evolution of the current protein-based system for replication and transcription? All nucleic acid polymerases are built from protein and all appear to use the same mechanism, namely two divalent metal ions that bridge across the a-phosphate of the incoming nucleotide triphosphate. The reaction catalyzed by the protein-based polymerase and the RNAbased intron are virtually equivalent. The nucleophile is the same in both cases, namely a 30 -OH (of the primer strand for polymerase, of the 50 -exon for splicing). The leaving group is different (G for the intron, pyrophosphate for the polymerase) but the organization of the metals is identical. For an RNA World to be viable, there must have been some kind of RNA-based polymerase to catalyze RNA replication. The group I structure demonstrates that RNA can fold and bind metals to create a polymerase-like active site, but it appears that biology has abandoned this catalyst. The reason for this transformation is likely to involve issues of fidelity, processivity, and reaction rates, all of which are expected to be greater in protein-based systems. Why hasn’t a similar selective pressure led protein to usurp RNA as the catalyst of peptide-bond formation? In spite of the fact that all nucleotide polymerases are made of protein, all amino acids are still polymerized within an RNA active site. The role of rRNA in this reaction appears to be largely organizational, binding to the CCA ends of each tRNA and positioning the reactive groups for chemistry. The key functional group is a 20 -OH located at the end of the tRNA, the substrate of the reaction. Under sufficient evolutionary pressure, protein should have taken over the role of aminoacyl polymerase just as it did for the nucleotidyl polymerase. If the issues www.sciencedirect.com
Acknowledgments Work described in this review was supported by NSF grant MCB-0544255 and NIH grants GM022778 and GM054839.
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26. Thompson J et al.: Analysis of mutations at residues A2451 and G2447 of 23S rRNA in the peptidyl transferase active site of the 50S ribosomal subunit. Proc Natl Acad Sci U S A 2001, 98:90029007. 27. Polacek N, Gaynor M, Yassin A, Mankin AS: Ribosomal peptidyl transferase can withstand mutations at the putative catalytic nucleotide. Nature 2001, 411:498-501. 28. Youngman EM, Brunelle JL, Kochaniak AB, Green R: The active site of the ribosome is composed of two layers of conserved nucleotides with distinct roles in peptide bond formation and peptide release. Cell 2004, 117:589-599. 29. Schmeing TM, Huang KS, Kitchen DE, Strobel SA, Steitz TA: Structural insights into the roles of water and the 20 hydroxyl of the P site tRNA in the peptidyl transferase reaction. Mol Cell 2005, 20:437-448. Complexes of the ribosomal peptidyl transferase center in complex with A-site and P-site substrates and a chiral transition state mimic reveal specifically bound waters and the proximity of the critical 20 -OH to the aamine and O30 leaving group. 30. Sievers A, Beringer M, Rodnina MV, Wolfenden R: The ribosome as an entropy trap. Proc Natl Acad Sci U S A 2004, 101:7897-7901. 31. Huang KS, Weinger JS, Butler EB, Strobel SA: Regiospecificity of the peptidyl tRNA ester within the ribosomal P site. J Am Chem Soc 2006, 128:3108-3109. 32. Weinger JS, Parnell KM, Dorner S, Green R, Strobel SA: Substrate-assisted catalysis of peptide bond formation by the ribosome. Nat Struct Mol Biol 2004, 11:1101-1106. 33. Erlacher MD et al.: Efficient ribosomal peptidyl transfer critically relies on the presence of the ribose 20 -OH at A2451 of 23S rRNA. J Am Chem Soc 2006, 128:4453-4459. Circularly permuted rRNA and ribosomal reconstitution makes it possible to perform atomic mutagenesis of functional groups in the peptidyl transferase center, including altering the 20 -OH of A2451, which is shown to be important for activity. 34. Dorner S, Polacek N, Schulmeister U, Panuschka C, Barta A: Molecular aspects of the ribosomal peptidyl transferase. Biochem Soc Trans 2002, 30:1131-1137. 35. Trobro S, Aqvist J: Mechanism of peptide bond synthesis on the ribosome. Proc Natl Acad Sci U S A 2005, 102:12395-12400. 36. Changalov M et al.: 20 /30 -O-Peptidyl adenosine as a general base catalyst of its own external peptidyl transfer: implications for the ribosome catalytic mechanism. Chem Biol Chem 2005, 6:992-996. 37. Rodnina MV, Beringer M, Wintermeyer W: Mechanism of peptide bond formation on the ribosome. Q Rev Biophys 2006, 39:203-225. 38. Woese CR: Translation: in retrospect and prospect. RNA 2001, 7:1055-1067. 39. Cochella L, Green R: An active role for tRNA in decoding beyond codon:anticodon pairing. Science 2005, 308:1178-1180. A conformational spring in the tRNA is implicated in the mechanism of induced fit, suggesting another active role for the tRNA substrates during translation. 40. Winkler WC, Nahvi A, Roth A, Collins JA, Breaker RR: Control of gene expression by a natural metabolite-responsive ribozyme. Nature 2004, 428:281-286. 41. McCarthy TJ et al.: Ligand requirements for glmS ribozyme self-cleavage. Chem Biol 2005, 12:1221-1226. www.sciencedirect.com
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42. Mandal M, Breaker RR: Gene regulation by riboswitches. Nat Rev Mol Cell Biol 2004, 5:451-463. 43. Hampel KJ, Tinsley MM: Evidence for preorganization of the glmS ribozyme ligand binding pocket. Biochemistry 2006, 45:7861-7871. 44. Klein DJ, Ferre-D’Amare AR: Structural basis of glmS ribozyme activation by glucosamine-6-phosphate. Science 2006, 313:1752-1756. X-ray crystal structure of the glmS ribozyme bound to a competitive inhibitor implicates the catalytic cofactor in the chemical mechanism of strand scission by the ribozyme.
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45. Cochrane JC, Lipchock SV, Strobel SA: Structural investigation of the GlmS ribozyme bound to its catalytic cofactor. Chem Biol 2007, 14:97-105. Structure of the glmS ribozyme bound to the GlcN6P activator and biochemical data on cofactor activation implicate GlcN6P as a general acid in ribozyme catalysis. 46. Selmer M et al.: Structure of the 70S ribosome complexed with mRNA and tRNA. Science 2006, 313:1935-1942. 47. Maguire B, Beniaminov A, Ramu H, Mankin A, Zimmermann R: A protein component at the heart of an RNA machine: the importance of protein L27 for the function of the bacterial ribosome. Mol Cell 2005, 20:427-435.
Current Opinion in Chemical Biology 2007, 11:636–643
Current Opinion in Chemical Biology 2007, 11:636–643 This review comes from a themed issue on Biopolymers Edited by Jennifer Kohler and Jason Chin
In this review, we will examine four examples of RNA catalysis, each of which provides a different chemical strategy used to promote a ribozyme reaction. This includes: the group I class of self-splicing introns which employs two divalent metal ions to promote RNA cleavage and ligation reactions; the Hepatitis Delta Virus ribozyme which catalyzes autolytic RNA cleavage using an essential, pKa perturbed cytidine; the glmS ribozyme which is an autolytic riboswitch that uses the small molecule metabolite glucosamine-6-phosphate (GlcN6P) as a catalytic cofactor; and the ribosomal peptidyl transferase center which does not appear to use any of these strategies, but instead forms peptide bonds with the assistance of a functional group on the tRNA substrate. The diversity of solutions employed by RNA to achieve reaction catalysis is unexpected; raising intriguing questions about the evolution and devolution of RNA catalysis. What led RNA to lose its catalytic role in some biological cases but not others, and what other catalytic functions might RNA still be playing in biology?
Available online 5th November 2007 1367-5931/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2007.09.010
RNA is well suited for its role as a purveyor of genetic information. The consistent hydrogen-bonding potential of each nucleobase is crucial for fidelity during replication, transcription, and translation. It is easy to imagine evolutionary pressures that would select for residues with such properties. This is achieved in part because none of the nucleobases have ionizable groups near neutrality. Although optimized for reliable base pairing, RNA is also able to catalyze essential biochemical reactions, including RNA processing and protein synthesis. It does this despite significant biophysical handicaps. RNA has a small repertoire of functional groups and these are embedded in a poorly constrained ribosyl-phosphate backbone heavy with negative charge. The pKas of the bases would appear to be either too low (3.5 for A and 4.2 for C) or too high (9.8 for G and 10.5 for U) for use in efficient general acid catalysis or base catalysis. As a group, RNA enzymes, or ribozymes, are less efficient catalysts relative to chemically supercharged proteins, yet in many cases ribozymes provide enough rate enhancement to have escaped replacement by protein alternatives in the evolution from an RNA World to the protein dominated world of modern biochemistry. Current Opinion in Chemical Biology 2007, 11:636–643
Group I intron — two-metal mechanism RNA splicing by the group I intron involves two symmetrical phosphoryl transfer reactions, both catalyzed by the intron itself (Figure 1). In the first reaction, an exogenous G attacks the 50 -splice site to release the 50 exon from the intron. In the second reaction, the 50 -exon attacks the 30 -splice site, at the phosphate 30 to the last G of the intron (VG), to produce ligated exons and liberate the intron. The second reaction is essentially the reverse of the first, differing only in whether exogenous G or VG is bound in the active site. An early model for group I intron catalysis was proposed by Steitz and Steitz based upon analogy to protein enzymes that catalyze phosphoryl transfer [1]. Their proposal involved two active site divalent metal ions bridging across the scissile phosphate positioned 3.9 A˚ apart, one activating the O30 nucleophile and the other activating the O30 leaving group. Although at the time no evidence for this model was provided, it was greeted enthusiastically and efforts were made to find these catalytic metals within the group I intron and other ribozymes. Biochemical analysis provided the initial evidence for metal ion participation in group I intron catalysis. Active site metal ion coordination was probed using metal specificity switch analysis. These experiments change the identity of a putative metal ligand from an oxygen to a sulfur. The electronically ‘soft’ sulfur substitution disrupts Mg2+ coordination, rendering the RNA inactive when the substitution is at a site of metal binding. Activity rescue upon addition of a ‘soft’ metal provides www.sciencedirect.com
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Figure 1
Group I intron splicing promoted by two catalytic metal ions. The second of the two-step splicing reactions is shown. The nucleophile (U-1 O30 ), scissile phosphate, leaving group (VG O30 ), and labile bond are shown in orange. Both catalytic metal ions are shown in green. A similar coloring scheme is used in all four figures where the chemically reactive groups and labile bond are shown in orange and the catalytically important groups are shown in green. (a) Active site structure adapted from Ref. [7]. (b) Reaction mechanism [7].
evidence for metal ion coordination. Single and pair wise substitutions were made throughout the group I intron active site. Consistent with the Steitz and Steitz model, the biochemical data suggested that metals coordinate the nucleophile, leaving group and scissile phosphate, but the intron also has a metal coordinating the 20 -OH vicinal to the leaving group (Figure 1). As a result, an alternative three-metal mechanism was proposed, in which none of the metals bridged between the scissile phosphate and the VG O30 leaving group [2,3]. This active site architecture differed significantly from that proposed for the two-metal model. X-ray crystal structures of the group I intron became available in the past few years. Structures of three different www.sciencedirect.com
introns in three different reaction states were reported in 2004 [4–6]. These provided three-dimensional views of the group I intron, but the RNA in each of these structures lacked at least one of the active site functional groups known to coordinate a catalytic metal ion. The structure of a catalytically active complex, including the complete intron and both exons and all metal ligands, was reported a year later (Figure 1) [7]. The overall structure of the intron was unchanged from the previous structures, but significant differences were observed within the active site. The structure revealed two well-ordered and extensively coordinated Mg2+ ions positioned 3.9 A˚ apart, both making inner sphere coordination to the scissile phosphate. Metal M1 is coordinated to the terminal O30 of the 50 -exon and the pro-RP oxygen of the scissile phosphate. Metal M2 also makes inner sphere coordination to the pro-RP oxygen of the scissile phosphate, as well as the O30 and O20 of VG. Both metals have a total of five inner sphere coordinations including interactions with phosphate oxygens within the intron. The two metals are coordinated by all of the biochemically predicted ligands, including two ligands identified within the intron. This two-metal ion structure suggests that the group I intron active site is mechanistically equivalent to a large number of protein-based phosphoryl transferases, including all known DNA and RNA polymerases. RNA enzymes and protein enzymes could not be evolutionarily related, so the equivalence of group I intron and polymerase active sites must result from convergent evolution. The intron uses the phosphate backbone in a manner equivalent to aspartate site chains in proteins for active site metal ion coordination, including one phosphate within the intron that uses both of its nonbridging oxygens to directly coordinate both active site metals. That macromolecular evolution arrived independently at the same solution in RNA and proteins implies an intrinsic catalytic capacity of the two-metal-ion catalytic architecture for phosphoryl transfer. What remains unknown is whether other RNA enzymes, such as the group II intron, RNase P, or the spliceosome, use the same catalytic strategy. Two RNase P structures were reported recently, but both complexes are of apoenzymes without the tRNA substrate [8,9]. Biochemical experiments strongly implicate metal ions in catalysis [10], but conformational changes or metal ion rearrangements might accompany substrate binding. No highresolution structural views of the group II intron or splicesome exist, however biochemical data implicate divalent metal ions in catalysis in both systems [11,12].
HDV ribozyme — nucleobase catalysis Divalent metals are not the only solution RNA has found to catalyze its reactions. The symmetry of the two-metal mechanism led to the premature conclusion that all ribozymes are metalloenzymes. Not every protein enzyme Current Opinion in Chemical Biology 2007, 11:636–643
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uses metals for phosphoryl transfer, and it is now clear that this is also true for ribozymes. For example, ribonuclease A employs two catalytic histidines to activate the nucleophile and stabilize the leaving group during the RNA cleavage reaction it promotes. Histidine, with its pKa of 6.8, is ideally suited for these roles at physiological pH. Because RNA lacks any nucleobase functional groups with a pKa near neutrality, nucleobase participation appeared to be an unlikely contributor to ribozyme chemistry. But it was then observed that several catalytic RNAs, including the HDV ribozyme, retained reasonable activity in the presence of high concentrations of monovalent ions, or other polyvalent ions that could not make inner sphere coordination to RNA ligands [13]. This suggested that alternative mechanisms must be in play. The HDV ribozyme is a small autonucleolytic RNA that shares a common chemistry with the other nucleolytic ribozymes. The 20 -OH next to the scissile phosphate acts as nucleophile to displace the O50 resulting in two RNA products, one with a 20 –30 cyclic phosphate terminus and one with a 50 -OH terminus (Figure 2). This is the same chemical reaction catalyzed by ribonuclease A. The first HDV crystal structure, of the product form of the ribozyme, showed that the 50 -OH was within hydrogen-bonding distance to the N3 of C75, an invariant residue indispensable for efficient cleavage [14]. The inactive C75U mutation could be rescued with imidazole and the pKa of the reaction appeared to track with the pKa of nucleobases substituted at the key residue [15,16]. This suggested that C75 might function as a general acid to protonate the O50 leaving group. However, subsequent crystal structures of the substrate form of the ribozyme showed that the C75 N3 is closer to the nucleophile, instead implicating C75 as a general base to deprotonate the 20 -OH [17]. This controversy was biochemically resolved in an elegant experiment showing that an enzyme with an activated leaving group no longer required this C for function [18]. These data strongly favor the original model; C75 acts as a general acid to protonate the leaving group and a divalent metal ion activates the nucleophile (Figure 2). Although C75 is not as efficient as histidine in this role because of its acidic pKa, the microenvironment of the enzyme active site appears to perturb the pKa toward neutrality. The precedent of general acid catalysis by the HDV ribozyme argues that other catalytic RNAs could employ such a strategy. Nucleobase catalysis has now been implicated in all of the autolytic ribozymes, but the diversity of the bases involved has come as a surprise. A critical A has been implicated in the VS ribozyme [19]. The crystal structure of the full-length hammerhead ribozyme reveals two critical Gs in the active site [20]. The N1 of G12 is near the O20 nucleophile and the 20 -OH of G8 is close to the O50 leaving group. The participation of G in RNA catalysis is unexpected as it has Current Opinion in Chemical Biology 2007, 11:636–643
Figure 2
Autolytic cleavage of RNA by the HDV ribozyme. Color scheme as in Figure 1. (a) Active site structure adapted from Ref. [14]. Residue A-1 was modeled into the coordinates for ease of comparison to the chemical scheme. (b) Reaction mechanism [16,18].
a alkaline pKa and the deprotonated form is anionic. It is relatively easy to envision how the highly anionic environment of an RNA could perturb a cationic pKa higher, but its harder to understand how the electrostatics of RNA could perturb an anionic pKa lower. Yet, Gs are also implicated in catalysis by the hairpin, VS and glmS (see below) ribozymes [21,22]. Proposed models for the role these Gs play in the reaction include simple substrate alignment by hydrogen bonding or proton transfer through tautomerization of the base [23,24].
Ribosomal peptide-bond formation by substrate assistance The peptidyl transferase center of the ribosome is also a ribozyme [25]. Initial mechanistic models for this reaction employed nucleobase involvement in catalysis (specifically A2451), however mutational analysis of this and www.sciencedirect.com
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other active site residues found that they contributed relatively little to the rate of peptide-bond formation [26– 28]. The effects were larger for the hydrolysis reaction that occurs at termination, but even in that reaction the largest rate decreases were still about 100-fold. Further, all crystallographic efforts to identify active site metal ions have also been unsuccessful [29]. While there is an obligate Mg2+ requirement for peptidyl transferase activity, the contribution does not appear to result from direct chemical participation. Relative to the rate in solution, the peptidyl transferase center of the ribosome provides a rate enhancement of approximately 107-fold, a contribution that has been largely attributed to entropy [30]. Beyond substrate alignment, how else might the ribosome promote this biologically essential reaction? The peptidyl transferase reaction involves two substrate tRNAs, an aminoacyl-tRNA that binds to the ribosomal A site and a peptidyl tRNA that binds to the P site. The nascent peptide in the P site is linked via an ester to the 30 -O of the terminal nucleoside of the tRNA, A76 Figure 3
(Figure 3) [31]. The reaction involves aminolysis of the P-site ester by the A-site a-amino group. The ribosome aligns the two substrates such that the amine approaches the sc face of the P-site ester, resulting in a chiral transition state with an S stereochemistry [29]. The A76 20 -OH vicinal to the P-site ester is essential for the reaction [32]. Deletion or alteration of this functional group results in a complete loss of peptidyl transferase activity (>106-fold loss of activity). Without this hydroxyl group, the rate of spontaneous ester hydrolysis is faster than the rate of peptide-bond formation. This contribution is significantly greater than that made by any of the rRNA nucleobases within the active site, though the 20 -OH of A2451 has also been shown to contribute to the reaction [33]. Hydroxyl groups are unlikely catalytic players in a reaction that appears to involve proton transfer. With a pKa of approximately 12, the 20 -OH is probably less likely than the nucleotide bases to be involved in proton transfer even if the pKa were significantly perturbed. Furthermore, there is nothing about the ribosome active site that would lead to such a perturbation. All unexplained density near the reaction center appears to be water, including one molecule positioned within the oxyanion hole [29]. Mechanistic models must therefore explain the role of the 20 -OH without invoking a significant degree of protonation or deprotonation. The ribosome positions the critical A76 20 -OH between the a-amino nucleophile and the 30 -O leaving group (Figure 3). As the amine must lose a proton and the O30 must gain one, mechanisms have been proposed that involve concerted proton transfer from the a-amine to the A76 O30 via the A76 20 -OH [29,34–37]. These mechanisms explain the need for the 20 -OH without having it assume a significant charge, consistent with its elevated pKa. Several mechanisms have been proposed that invoke this role for the 20 -OH, each differing in the extent of peptide-bond formation and the degree of leaving group dissociation in the transition state. Although the mechanisms share common themes, they make significantly different predictions regarding the nature of the transition state and the contribution the ribosome could make to catalysis. The answer will require enzymatic analysis to define the degree of bonding and distribution of charge in the transition state. Only then will it be clear exactly what transition state the ribosome stabilizes during peptidebond formation and the precise role of the 20 -OH of A76.
Peptide-bond formation promoted by the ribosome. The a-amine, carbonyl carbon, O30 leaving group and scissile bond are shown in orange. The critical 20 -OH is in green. (a) Active site structure adapted from Ref. [29]. (b) Proposed reaction mechanism [29,34]. www.sciencedirect.com
Independent of these mechanistic details, the observation that a functional group on the tRNA substrate provides a major catalytic contribution invokes an additional role for tRNAs in translation. Even before the discovery of tRNA, Crick proposed the adaptor hypothesis, which predicted a molecule capable of two functions: interaction with an Current Opinion in Chemical Biology 2007, 11:636–643
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mRNA and delivery of the correct amino acid. tRNA plays both these roles, but the adaptor model suggests a passive role for tRNA during translation. tRNA is aminoacylated by synthases and used by the ribosome for decoding, but the tRNAs are the object, rather than the subject of action in these roles. In a recent review, Woese called for a reevaluation of tRNA during translation [38], and there are now indications that tRNA does play a significant proactive role, including a spring-like distortion of the tRNA during accommodation into the A site where it provides the key functional group for peptide-bond formation to proceed [32,39].
Figure 4
GlmS ribozyme — use of a catalytic cofactor And when it appeared that all permutations for RNA catalysis had been exhausted, a ribozyme was identified that uses still another mechanistic strategy (Figure 4). The glmS riboswitch is located in the 50 -untranslated region of the gene encoding glucosamine-6-phosphate (GlcN6P) synthetase [40]. In the presence of GlcN6P, it cleaves its own mRNA, which downregulates the production of the synthetase. GlcN6P is essential for efficient cleavage activity, though other primary amines with a vicinal hydroxyl group can also activate the ribozyme in vitro, including serinol and Tris [41]. The primary amine is essential for the reaction; substitution of the amine with a hydroxyl results in complete loss of activity. Glucose-6 phosphate (Glc6P) is still able to bind to the ribozyme, but functions as a competitive inhibitor. Two possible models for the role of GlcN6P in glmS catalysis can be envisioned. In traditional riboswitches, metabolite binding induces a conformational change in the RNA that affects the transcription or translation of the gene [42]. In this model for riboswitch function, binding of the GlcN6P would stabilize the active ribozyme conformation and lead to catalysis. In the alternative model, GlcN6P binding does not affect the conformation of the RNA, but instead the critical amine on the metabolite participates directly in chemistry. Biochemical and structural data both indicate that the contribution made by GlcN6P is not conformational, but is instead chemical [40,43,44,45]. Initial inline probing experiments showed that the conformational flexibility of the RNA is unchanged upon GlcN6P binding [40]. Hydroxyl radical footprinting experiments showed that the RNA had an equivalent solvent accessible surface in both the bound and free states [43]. Consistent with these observations, independent crystal structures revealed that the RNA adopts the same conformation in the metabolite bound, inhibitor bound and free states [44,45]. Thus, glmS is not a traditional riboswitch, changing its conformation upon metabolite binding, instead it uses the chemical capability of the metabolite in catalysis. The pKa of the GlcN6P amine is 8.3, a value reasonably well suited for a role in proton transfer. This demonstrates that Current Opinion in Chemical Biology 2007, 11:636–643
Autolytic cleavage of RNA by the glmS ribozyme in the presence of GlcN6P. The color scheme is as in Figure 1 with the critical amine of GlcN6P and the N1 of G33 shown in green. (a) Active site structure adapted from Ref. [45]. (b) Proposed reaction mechanism [45]. The identity of G33 is important to the reaction, but its role remains to be fully defined.
RNA, like protein enzymes, can employ the chemical potential of small molecules to promote catalytic activity. How does GlcN6P serve as a catalytic cofactor? The authors of the initial structure of the Glc6P inhibitor bound ribozyme suggested that the amine might serve as a general base to deprotonate the O20 nucleophile [44]. Although the C2 hydroxyl is not well positioned for this role, the authors proposed that it could act through a chain of two water molecules present in the active site. The GlcN6P bound structure superimposes on the Glc6P structure, but no water molecules for proton shuttling to the O20 were observed [45]. In both structures the amine (or its hydroxyl substitute) is within hydrogenbond distance of the O50 leaving group (Figure 4). This suggests a role for the amine as a general acid to activate www.sciencedirect.com
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the leaving group. A definitive answer to this question awaits the kind of analysis that was performed on the HDV ribozyme [18]. The closest functional group to the O20 nucleophile is not on GlcN6P, but is the N1 of G33 (Figure 4). Consistent with a role in ribozyme function, mutation of G33 disrupts activity [45]. However, the precise contribution of this functional group to glmS ribozyme catalysis is also unknown. Nevertheless, the primary catalytic contribution is expected to come through the metabolic cofactor, as activation of the leaving group is the chemically difficult step in phosphoryl transfer reactions.
are fidelity, processivity, and reaction rates, why did these same parameters not lead to a protein-based system for peptide-bond formation? This has not happened, or at least hasn’t happened yet. The amino terminus of protein L27 is intriguingly close to the peptidyl transferase center as observed in the recent 70S crystal structure from Thermus thermophilus [46]. L27 is not conserved and so is not essential for protein synthesis in all organisms, but this terminal extension is essential in E. coli [47]. L27 may represent an adaptation, possibly an evolutionary intermediate between an RNA and a protein-based active site for protein synthesis.
Implications
Studies of the glmS ribozyme revealed that RNA can use small molecules as catalytic cofactors. With an expanded chemical repertoire made possible by small organic compounds, what other reactions did (or still does) RNA catalyze in biological systems? To date, riboswitches responsive to at least 10 different small molecules have been identified [42]. The metabolites include coenyzme B12 (B12), flavin mononucleotide (FMN), thiamine pyrophosphate (TPP), S-adenosyl methionine (SAM), guanine, adenine, glycine, lysine, and GlcN6P. The intriguing nature of this list is that many of the metabolites that are recognized by riboswitches are also among the most ubiquitous cofactors used by protein enzymes, particularly B12, FMN, TPP, and SAM. Protein enzymes use these cofactors and substrates for radical chemistry, oxidation– reduction reaction and carbon–carbon bond formation. To date, chemistry promoted by naturally occurring RNAs has been confined to phosphoryl transfer and transesterification. Naturally occurring RNAs bind these small molecule cofactors. The use of their chemical moieties in chemistry could significantly expand the catalytic repertoire of RNA. Ribozymes with radical, redox and methyltransferase activities were needed for metabolism in the RNA World. Which, if any of them, have persisted into modern biology remains to be determined.
The mechanisms employed by RNA to promote chemistry are substantially more diverse than once thought. They include metals, nucleobases, ribosyl groups, and even small molecule cofactors. Such observations raise questions regarding the evolution of biocatalysis and the diversity of reactions that RNA might catalyze. The observation that the group I intron has a polymeraselike active site built of RNA leads to the question: What selective pressure led to the evolution of the current protein-based system for replication and transcription? All nucleic acid polymerases are built from protein and all appear to use the same mechanism, namely two divalent metal ions that bridge across the a-phosphate of the incoming nucleotide triphosphate. The reaction catalyzed by the protein-based polymerase and the RNAbased intron are virtually equivalent. The nucleophile is the same in both cases, namely a 30 -OH (of the primer strand for polymerase, of the 50 -exon for splicing). The leaving group is different (G for the intron, pyrophosphate for the polymerase) but the organization of the metals is identical. For an RNA World to be viable, there must have been some kind of RNA-based polymerase to catalyze RNA replication. The group I structure demonstrates that RNA can fold and bind metals to create a polymerase-like active site, but it appears that biology has abandoned this catalyst. The reason for this transformation is likely to involve issues of fidelity, processivity, and reaction rates, all of which are expected to be greater in protein-based systems. Why hasn’t a similar selective pressure led protein to usurp RNA as the catalyst of peptide-bond formation? In spite of the fact that all nucleotide polymerases are made of protein, all amino acids are still polymerized within an RNA active site. The role of rRNA in this reaction appears to be largely organizational, binding to the CCA ends of each tRNA and positioning the reactive groups for chemistry. The key functional group is a 20 -OH located at the end of the tRNA, the substrate of the reaction. Under sufficient evolutionary pressure, protein should have taken over the role of aminoacyl polymerase just as it did for the nucleotidyl polymerase. If the issues www.sciencedirect.com
Acknowledgments Work described in this review was supported by NSF grant MCB-0544255 and NIH grants GM022778 and GM054839.
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